Physiological Calcium Concentrations Regulate Calmodulin Binding and Catalysis of Adenylyl Cyclase Exotoxins

The EMBO Journal Vol. 21 No. 24 pp. 6721±6732, 2002 Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins Yuequan Shen1, Young-Sam Lee2, the central helix and the calcium-induced changes in Sandriyana Soelaiman1, Pamela Bergson1,3, surface properties enable CaM to bind and modulate a Dan Lu1, Alice Chen1, Kathy Beckingham4, diverse array of physiologically important proteins, such Zenon Grabarek5, Milan Mrksich2 and as adenylyl cyclase, phosphodiesterase, nitric oxide Wei-Jen Tang1,3,6 synthase, protein kinase and phosphatase, receptor and ion channel (Eldik and Watterson, 1998; DeMaria et al., 1Ben-May Institute for Cancer Research, 2Department of Chemistry, 2001). Consequently, CaM is involved in many intra- 3 and Committee on Neurobiology, The University of Chicago, cellular processes including control of transcription, ion Chicago, IL 60637, 4Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251 and 5Boston Biomedical ¯uxes, signal transduction, vesicular transport and cyto- Research Institute, Watertown, MA 02472, USA skeleton functions (Deisseroth et al., 1998; Eldik and Watterson, 1998). 6Corresponding author e-mail: [email protected] Structural and biochemical analyses have provided insights into CaM-dependent regulation of some target Edema factor (EF) and CyaA are calmodulin (CaM)- enzymes (Hoe¯ich and Ikura, 2002; Meador and Quiocho, activated adenylyl cyclase exotoxins involved in the 2002). The best-known activation mechanism is the pathogenesis of anthrax and whooping cough, respect- release of autoinhibition exempli®ed by myosin light ively. Using spectroscopic, enzyme kinetic and surface chain kinase (MLCK) and CaM kinase II (CaMKII). In this plasmon resonance spectroscopy analyses, we show model, CaM is not associated with the target enzyme at the that low Ca2+ concentrations increase the af®nity of resting Ca2+ levels, and the enzyme's catalytic activity is CaM for EF and CyaA causing their activation, but blocked by its own autoinhibitory domain (AID). Upon higher Ca2+ concentrations directly inhibit catalysis. increase in the intracellular Ca2+ concentration, the Both events occur in a physiologically relevant range Ca2+±CaM complex binds to an amphipathic a-helix that of Ca2+ concentrations. Despite the similarity in Ca2+ partially overlaps the AID. This presumably causes a sensitivity, EF and CyaA have substantial differences conformational change that disrupts the interaction of the in CaM binding and activation. CyaA has 100-fold AID with the catalytic domain, resulting in kinase higher af®nity for CaM than EF. CaM has N- and activation. A recent structure of the complex of CaM C-terminal globular domains, each binding two Ca2+ with the intracellular domain of the small conductance 2+ ions. CyaA can be fully activated by CaM mutants Ca -activated potassium channel, Ik(Ca), reveals a new with one defective C-terminal Ca2+-binding site or by activation mechanism (Schumacher et al., 2001). In this either terminal domain of CaM while EF cannot. EF case, CaM is constitutively bound to Ik(Ca). Calcium consists of a catalytic core and a helical domain, loading allows CaM to act as a clamp to induce Ik(Ca) and both are required for CaM activation of EF. dimerization, which leads to an allosteric change and Mutations that decrease the interaction of the helical increased ion conductivity. domain with the catalytic core create an enzyme with Several pathogenic bacteria, such as those that cause higher sensitivity to Ca2+±CaM activation. However, anthrax (Bacillus anthracis), whooping cough (Bordetella CyaA is fully activated by CaM without the domain pertussis) and cholera (Vibrio cholerae), secrete toxins corresponding to the helical domain of EF. that increase the cAMP concentration in the host cells to a Keywords: adenylyl cyclase exotoxin/anthrax edema pathological level (Drum et al., 2002). Edema factor (EF) factor/Ca2+±calmodulin/CyaA/enzyme activation and CyaA are adenylyl cyclase toxins produced by B.anthracis and B.pertussis, respectively (Ladant and Ullmann, 1999; Mock and Fouet, 2001). Both EF and CyaA are activated in the host cell by CaM. EF is Introduction responsible for the massive edema seen in cutaneous Calcium serves as a diffusible second messenger in anthrax and impairs the function of neutrophils and response to extra- and intracellular signals, and calmodulin monocytes in systemic infection (Hoover et al., 1994). (CaM) is a key calcium sensor (Eldik and Watterson, CyaA is important for the bacterial colonization of the 1998). CaM has two globular domains, each consisting of respiratory tract, in part owing to its ability to induce two helix±loop±helix calcium-binding motifs (Babu et al., apoptosis of macrophages (Khelef et al., 1993; Weingart 1985). The domains are linked by a ¯exible a-helix that is and Weiss, 2000). partially unfolded in solution (Barbato et al., 1992). The CaM-activated adenylyl cyclase domain resides in Calcium induces a transition in both domains from a the C-terminal 510 amino acid region of EF. The closed conformation with highly negatively charged molecular structures of this domain with and without surface to an open conformation with a large, exposed CaM were solved recently. These structures have provided hydrophobic pocket (Finn et al., 1995). The ¯exibility of a new model of CaM binding and activation of a target ã European Molecular Biology Organization 6721 Y.Shen et al. Fig. 1. The effect of calcium ions and CaM on the adenylyl cyclase activities of EF and CyaA-N. Adenylyl cyclase assays were performed in the presence of 1 nM EF (A) and 0.7 nM CyaA-N (B) under 10 mM CaM (®lled circles), 0.1 mM CaM (open circles) and 1 nM CaM (®lled triangles, CyaA-N only) at increasing [Ca2+]. They were also performed at 0.1 mMCa2+ (®lled circles), 0.3 mMCa2+ (open circles) and 1.0 mMCa2+ (®lled triangles) at increasing [CaM] in the presence of 1 nM EF (C), and 0.7 nM CyaA-N (D). Maximal adenylyl cyclase activities (100%) for EF in the calcium titration are 1140 s±1 (10 mM CaM) and 228 s±1 (0.1 mM CaM) (A) and those for CyaA-N are 1465 s±1 (10 mM CaM), 713 s±1 (0.1 mM CaM) and 556 s±1 (1 nM CaM) (B). Means 6 SE are representative of at least two experiments. enzyme (Drum et al., 2002; Hoe¯ich and Ikura, 2002; In this paper, we show that the physiological intra- Meador and Quiocho, 2002). Unlike the CaM complexes cellular calcium concentration not only dramatically with MLCK and CaMKII, in which CaM takes a compact increases the CaM af®nity of both EF and CyaA, form, in the complex with EF, CaM has an extended enhancing the activation of adenylyl cyclase activity, but conformation and makes extensive contacts (~6000 AÊ 2) also directly interferes with binding of the catalytic metal, with four discrete regions of EF. This interaction induces a inhibiting catalysis. We also show that the interaction of 15 AÊ translation and 30° rotation of a 15 kDa helical the helical domain of EF with CaM plays a key role in EF domain of EF resulting in stabilization of a 12 amino acid- activation. long loop. This loop contains several amino acid residues crucial for catalysis, and it is disordered in the structure of EF alone so that the catalytic site is open and incomplete. Results CaM-induced conformational changes stabilize this loop Physiological calcium and CaM concentrations are to enclose and complete the catalytic site, achieving over required for the optimal activation of EF 1000-fold enhancement in the catalytic rate of EF. The activation of EF by CaM can be greatly reduced by the There is considerable cross talk between the signaling addition of EGTA, a calcium chelator (Leppla, 1984). pathways of calcium and cAMP, two key intracellular However, it is not clear whether physiological concentra- messengers. An increase in intracellular cAMP levels can tions of calcium could modulate the activation of EF by elevate intracellular Ca2+ by the activation of calcium- CaM. While the estimated total CaM concentration inside permeable channels, such as L-type calcium channels, cells is ~1±10 mM, free CaM concentration is signi®cantly nicotinic acetylcholine receptors and cyclic nucleotide lower since it is associated with apo-CaM-binding proteins gated channels (Cooper et al., 1995). On the other hand, such as GAP-43 and RC3 (Gerendasy, 1999; Jurado et al., Ca2+±CaM can decrease the cAMP level by activation of 1999). We therefore measured adenylyl cyclase activity of phosphodiesterase, or increase it by activating speci®c EF at a broad range of calcium concentrations (1 nM to isoforms of adenylyl cyclase (type I, III and VIII; Eldik 10 mM) in the presence of either 0.1 or 10 mM CaM, and Watterson, 1998; Hanoune and Defer, 2001). Little is representing the low and high ends of the intracellular free known regarding the effects of physiological Ca2+ con- CaM concentration range (Figure 1A). We found that, centrations on the adenylyl cyclase activity of EF and at both CaM concentrations, adenylyl cyclase activity CyaA. This question is especially intriguing in view of the exhibited a bell-shaped curve with optimal activity at the fact that Ca2+ ions were found only in the C-terminal physiologically relevant calcium concentrations (0.1± domain of CaM in the structure of EF±CaM complex. 0.5 mM). While <5-fold change in the adenylyl cyclase 6722 Activation of EF and CyaA by Ca2+±calmodulin activity of EF at the 0.03±10 mM calcium concentrations anthraniloyl group in 2¢d3¢ANT-ATP is covered by a was observed in the presence of 10 mM CaM, the catalytic loop called switch B (Figure 2C and D). Switch B enzymatic activity of EF was sharply altered by the is not visible in the EF alone structure, but it is stabilized same calcium concentrations when 100-fold less CaM by switch C and becomes ordered upon CaM binding.

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